Separation of the complexes formed between the regulatory and catalytic subunits of cyclic adenosine monophosphate-dependent protein kinase and topoisomerase I activity in preovulatory follicle-enriched immature rat ovaries

Abstract

Our previous studies have shown that the regulatory subunits of the type II form of cAMP-dependent protein kinase (R‖) present in soluble extracts of immature rat ovaries elute from diethylaminoethyl-cellulose as three separate peaks of activity, based on their association with the catalytic subunit (C) of this enzyme, as R2 ‖C2, an apparent R2 ‖C, and R2 ‖. Based upon the existence of ovarian R‖ in three different subunit arrangements, the large amount of C subunit-free R2 ‖ in this tissue, and a previous report which indicated that R‖ exhibited intrinsic topoisomerase I activity, we determined whether DNA topoisomerase I activity was associated with any of these molecular complexes of the ovarian R‖ subunits. Cyclic AMP-binding activities in soluble extracts of preovulatory follicle-enriched immature rat ovaries were separated by diethylaminoethyl-cellulose chromatography and sucrose density gradient centrifugation. Topoisomerase I activity cosedimented with the apparent R2 ‖C and R2 ‖ obtained from sucrose gradients but was not detected in fractions containing R2 ‖C2. Upon cAMP affinity purification of the R‖ derived from fractions containing R2 ‖C2, R2 ‖C, and R2 ‖, respectively, no topoisomerase I activity could be detected in any fraction. Phosphorylation of the affinity purified R‖s by the C subunit of beef heart cAMP-dependent protein kinase did not alter this result. These data indicate that none of the R‖ subunits in soluble extracts of preovulatory follicle-enriched ovaries exhibit intrinsic topoisomerase I activity. © 1989 by The Endocrine Society.